AGRO/ANSC/BIO/GENE/HORT 305
Fall, 2017
Gene transcription and RNA modification
Chpt 12, Genetics by Brooker, Lecture Outline #10
Transcription is the first step in gene expression. In genetics, the term refers to the copying of a DNA sequence into RNA. It involves two fundamental concepts:
- DNA sequences provide the underlying information.
- sequences for the start and end of transcription.
- Proteins recognize these sequences and carry out the process
- Other proteins modify the RNA transcript to make it functionally active.
At the molecular level, a gene is a transcriptional unit
- the strand that is actually transcribed is called the template strand.
- The opposite strand is called the coding strand or the sense strand
o The base sequence of the coding strand is identical to the RNA sequence except for substitution of uracil in RNA for thymine in DNA.
- Three stages of transcription:
- Initiation
- Elongation
- Termination
These steps involve protein-DNA interactions: proteins such as RNA polymerase interact with DNA sequences.
- RNA transcripts have different functions
- A structural gene is one that encodes a polypeptide
o When such genes are transcribed, the product is an RNA transcript called messenger RNA (mRNA). Well over 90% of all genes are structural genes.
- The RNA transcripts from nonstructural genes are not translated
o They do have various important cellular functions.
o In some cases the RNA transcripts becomes part of a complex that contain protein subunits
§ Ribosomes
§ Spliceosomes
§ Signal recognition particles
§ Telomerase
Transcription in Bacteria:
- Promoters are DNA sequences that “promote” transcription; more precisely the exact location of transcription initiation.
Promoters are typically located just upstream of the site where transcription of a gene actually begins. The bases in a promoter sequence are numbered in relation to the transcription start site.
- RNA polymerase is the enzyme that catalyzes the synthesis of RNA
- In E.coli, the RNA polymerase holoenzyme is composed of
o Core enzyme – 4 subunits a2bb'w
o Sigma factor – 1 subunit s
- The RNA polymerase holoenzyme binds loosely to the DNA
It then scans along the DNA till it encounters the promoter region. When it does, the Sigma factor recognizes both -10 and -35 regions. A region within the Sigma factor that contains a helix-turn-helix structure is involved in the tighter binding to the DNA.
- The binding of the RNA polymerase to the promoter forms the closed complex. Then the open complex is formed when the TATAAT box is unwound. A short RNA strand is formed within the open complex. The Sigma factor is released at this point and this marks the end of initiation. The core enzyme now slides down the DNA to synthesize an RNA strand.
- The open complex formed by the action of the RNA polymerase is about 17 bases long. Behind the open complex, the DNA rewinds back into a double helix.
On average, the rate of RNA synthesis is 43 nucleotides per second.
- Termination is the end of RNA synthesis. It occurs when the short RNA-DNA hybrid in the open complex is forced to separate. This releases the newly made RNA and also the RNA polymerase.
E. coli has two different mechanisms for termination
o Rho-dependent termination- requires a protein r
o Rho-independent termination- does not require r
- Many of the basic features of gene transcription are very similar in bacteria and eukaryotes. However, gene transcription in the eukaryotes is more complex for the following reasons:
o Larger organisms
o Cellular complexity
o Multicellularity
- Nuclear DNA is transcribed by three different RNA polymerases
o RNA pol I: Transcribes all rRNA except 5S rRNA
o RNA pol II: Transcribes all structural genes and some snRNA genes
o RNA pol III: Transcribes all tRNA genes and 5S rRNA gene
- All three are very similar structurally and are composed of many subunits
- There is also a remarkable similarity between the bacterial RNA polymerase and its eukaryotic counterparts.
- For structural genes, at least three features are found in most promoters
o Transcriptional start sites, TATA box, Regulatory elements or cis-elements.
- The core promoter is relatively short and it consists of the TATA box. The TATA box determines the precise start point for transcription
- The core promoter by itself produces a low level of transcripts. This is termed basal transcription.
- Regulatory elements affect the binding of RNA polymerase to the promoter
o Enhancers: stimulates transcription
o Silencers: inhibit transcription
- They vary in their location but are often found in -50 to -100 region
- Factors that control gene expression can be divided into two types, based on their location:
- cis-acting elements: DNA sequences that exert their effect on genes that are contiguous.
- trans-acting elements/factors: Regulatory proteins that bind to the cis-elements
RNA Polymerase II and its transcription factors:
- three categories of proteins are required for basal transcription to occur at the promoter – Basal transcription apparatus
o RNA polymerase II
o 5 different general transcription factors
o a protein complex called mediator, it mediates interaction between RNA pol II and the various regulatory proteins
Chromatin Structure and transcription:
- the compaction of DNA to form chromatin can be an obstacle to the transcription process. Most transcription occurs during interphase.
o Then chromatin is found in the 30nm fibers that are organized into radial loop domains
o Within the 30nm fibers, the DNA is wound round the histone octamers to form nucleosomes
o The histone octamer is roughly 5X smaller than the complex of RNA pol II and the general transcription factors.
o The tight wrapping of DNA within the nucleosome inhibits the function of RNA pol
o To circumvent this problem the chromatin structure is significantly loosened during transcription
o Two common mechanisms alter chromatin structure
§ Covalent modification of histones; amino terminal ends of the histones are modified in various ways: Acetylation, phosphorylation, methylation.
§ ATP-dependent chromatin remodeling; the energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible.
RNA modification: In bacteria, the DNA sequence in the coding regions are the same as in the mRNA; this in turn corresponds to the sequence of amino acids in the polypeptide; this is termed the colinearity of gene expression
- Eukaryotic structural genes are not always colinear with their functional mRNA.
o Instead coding sequences, called exons, are interrupted by intervening sequences or introns
o Transcription produces the entire gene product
§ Introns are later removed or excised
§ Exons are connected together or spliced. This is termed RNA splicing
o Aside from splicing, RNA transcripts can be modified in several ways
§ Trimming of rRNA and tRNA transcripts
§ 5’capping and 3’ polyA tailing of mRNA transcripts (Table
Capping:
- In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNA (also as heterogeneous nuclear RNA, hnRNA)
- This RNA is altered by splicing and other modifications, before it leaves the nucleus
- Splicing in this case requires the aid of a multicomponent structure known as the spliceosome.
- Most mature mRNAs have a 7-methyl guanosine covalently attached at their 5’end; this event is known as capping. Capping occurs as the pre-mRNA is being synthesized by RNA pol II.
o The 7-methylguanosine cap structure is recognized by cap-binding proteins. Cap-binding proteins play roles in:
§ Movement of some RNAs into the cytoplasm
§ Early stages of translation
§ Splicing of introns
Tailing
- Most mature mRNAs have a string of adenine nucleotides at their 3’ends; this is termed polyA tail.
- The polyA tail is not encoded in the gene sequence, it is added enzymatically after the gene is completely transcribed
- There is clipping of the transcript 20 nucleotides downstream of a consensus sequence AAUAAA, where the polyA tail is added
- polyA tail is important in the stability of mRNA and the translation of the polypeptide.
Pre-mRNA Splicing:
The spliceosome is a large complex that splices the pre-mRNA; it is composed of several subunits known as snRNPs (pronounced ‘snurps’); snRNP contains small nuclear RNA and a set of proteins
- The consensus sequences for splicing are: 5’GU----A----AG 3’
- Phenomenon of alternative splicing: a pre-mRNA with multiple introns can be spliced different ways to generate mature RNAs with different combinations of exons.
- The biological advantage of alternative splicing is that multiple polypeptides can be derived from a single gene; this allows an organism to carry fewer genes in the genome.
- Non structural genes: RNA molecules such as ribosomal RNA and tRNA precursors are processed to smaller, functional molecules via cleavage steps.
RNA editing:
RNA editing changes the base sequence of an RNA after it has been synthesized .
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